PROJECT SUMMARY/ABSTRACT Non-alcoholic fatty liver disease (NAFLD), the most common chronic liver disease worldwide, is characterized by aberrant dysregulation of cellular energy mechanisms, including defective autophagy and mitochondrial function. Inhibiting general autophagy in models of NAFLD has produced contradictory results, resulting in both protection against and causation of hepatic steatosis and insulin resistance. However, the specific role of autophagy to selectively degrade mitochondria?the process known as mitophagy?has not been studied in the context of NAFLD development. Understanding the precise role of mitophagy in NAFLD, as well as the molecular drivers underlying autophagy-mediated mitochondrial dysfunction, will bring us closer to developing durable therapeutic strategies to combat this disease. The main goals of this project are to establish the function of dysregulated mitophagy in the development of NAFLD, and to determine how Polycystin-2 (PC2), a Ca2+- modulating protein on the endoplasmic reticulum (ER), works to promote hyper-activation of mitophagy through its interaction with Bcl-2 associated athanogene 2 (BAG2). This will allow for understanding of how observed mitochondrial morphological and functional alterations that occur in hepatocytes following the onset of NAFLD are altered by the up-regulation of these proteins. My interest in PC2 and BAG2 is based on published and preliminary data demonstrating 1) increased PC2 and BAG2 expression in human and murine livers with NAFLD, 2) BAG2 as an interacting partner of PC2 that promotes PTEN-induced putative kinase 1 (PINK1)-mediated mitophagy following PC2 up-regulation, and 3) protection against insulin resistance and hepatic steatosis in PC2 haploinsufficient mice fed a high fat diet (HFD). This proposal will examine how PC2 is up-regulated under stress to modulate mitophagy and mitochondrial function, as well as how PC2-null hepatocytes respond to HFD- induced NAFLD. These results will confirm PC2 as a stress response protein capable of regulating mitophagy and will help resolve the role of dysregulated autophagy in NAFLD development. The specific mechanisms responsible for up-regulation of PC2 and BAG2 to promote mitophagy will be determined by cellular and biochemical approaches investigating the cell stressors leading to transcriptional increases in PC2 mRNA, how increased PC2 affects BAG2 expression and subcellular localization, and how these proteins work to increase mitophagy through the well-established PINK1 pathway. Mitophagy in NAFLD development will be examined through mice fed normal diet or HFD with or without hepatocyte PC2 expression (Pkd2fl/fl;AlbuminCre- or Pkd2fl/fl;AlbuminCre+), and metabolic characterization of these mice will be performed through hyperinsulinemic- euglycemic clamps and ex vivo analyses examining mitophagy, mitochondrial function, hepatic lipid retention, and calcium signaling. Human liver samples will also be studied in depth for a relationship between PC2, BAG2 and disease development. The results of these studies will provide insight into the identification of novel targets to develop effective therapies for rescuing excess mitochondrial fragmentation and dysfunction in NAFLD.